Shear stress generated by blood flow is one of the most important physiological regulators of vascular tone. Flow stimulates vascular endothelial cell to release vasodilator factors that subsequently relax underlying smooth muscle, a response often known as flow-mediated dilation (FMD). In human coronary arterioles (HCA), FMD results from the release of two entirely independent dilator factors: nitric oxide (NO) in subjects without coronary artery disease (CAD), and reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2) derived from the mitochondrial electron transport chain, in CAD patients. However, it remains unsolved how shear induces the release of these two distinct relaxing factors. The present proposal will test a central hypothesis that the transient receptor potential vanilloid 4 (TRPV4) channel serves as a common mechanism for the release of two otherwise diversely regulated relaxing factors (NO and mitochondria-derived H2O2) responsible for FMD in the human coronary microcirculation. We further propose that the signaling cascade occurs within caveolae that host novel interactions between the plasma membrane and mitochondria. Studies will be conducted on isolated HCA and cultured endothelial cells using an integrated approach incorporating molecular biology, electrophysiology and fluorescence/electron imaging techniques with in vitro assessment of vessel reactivity. Genetically engineered mice will also be used to provide more definitive corroboration of the human data. Three specific aims are proposed. Aim 1 will determine whether FMD requires endothelial TRPV4 in HCA from patients with or without CAD. We will test the effects of pharmacological inhibition and siRNA downregulation of TRPV4 on flow-induced Ca2+ entry, ROS/NO release, and vasodilation in HCA from CAD and non-CAD subjects. In aim 2, we will examine whether endothelial TRPV4 channels are associated with caveolae and whether this association is essential for shear-induced TRPV4 activation. We will test three caveolae-associated signaling events contributing to TRPV4 activation: namely, TRPV4 translocation, caveolin-1 regulation, and phospholipase A2-epoxyeicosatrienoic acids activation. In aim 3, we will determine whether shear increases mitochondrial ROS through localized Ca2+ signaling involving caveolar TRPV4 and adjacent mitochondria and whether this process is negatively regulated by NO. The proposed research will, for the first time, link endothelial TRPV4, caveolae, and mitochondria as essential signaling components for FMD in humans. We expect the outcomes of this proposal will substantially increase our understanding of the intricate signaling mechanisms involved in FMD in the human coronary microcirculation, and may lead to new therapeutic targets for the treatment of CAD and/or other cardiovascular disorders. PUBLIC HEALTH RELEVANCE: This grant proposal is designed to study a novel signaling mechanism by which shear stress, a mechanical force generated by blood flow, causes blood vessel dilation in humans. Specifically we examine whether a calcium ion channel (TRPV4) located on the cell surface membrane of vascular endothelial cells serves an essential signaling component for shear-induced dilation in human coronary microvessels. Flow or shear- induced dilation is one of the most important regulators of vascular tone and regional blood flow. Therefore the findings of this proposal will importantly contribute to our understanding of how coronary blood flow is regulated in normal and disease states, and may lead to new therapeutic targets for the treatment of coronary artery disease and/or other cardiovascular disorders.